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J. Biol. Chem., Vol. 279, Issue 30, 31964-31972, July 23, 2004

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Human Immunodeficiency Virus Type 1 Gag Assembly through Assembly Intermediates^*

Yuko Morikawa, Toshiyuki Goto¶, and Fumitaka Momose

From the Kitasato Institute of Life Sciences, Kitasato University, Shirokane 5-9-1, Minato-ku, Tokyo 108-8641 and the ^¶College of Medical Technology, Kyoto University, Kawahara-cho 53, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan

Human immunodeficiency virus Gag protein self-assembles into spherical particles, and recent reports suggest the formation of assembly intermediates during the process. To understand the nature of such assembly intermediates along with the mechanism of Gag assembly, we employed expression in Escherichia coli and an in vitro assembly reaction. When E. coli expression was performed at 37 °C, Gag predominantly assembled to a high order of multimer, apparently equivalent to the virus-like particles obtained following Gag expression in eukaryotic cells, through the formation of low orders of multimer characterized with a discreet sedimentation value of 60 S. Electron microscopy confirmed the presence of spherical particles in the E. coli cells. In contrast, expression at 30 °C resulted in the production of only the 60 S form of Gag multimer, and crescentshaped structures or small patches with double electron-dense layers were accumulated, but no complete particles. In vitro assembly reactions using purified Gag protein, when performed at 37 °C, also produced the high order of Gag multimers with some 60 S multimers, whereas the 30 °C reaction produced only the 60 S multimers. However, when the 60 S multimers were cross-linked so as not to allow conformational changes, in vitro assembly reactions at 37 °C did not produce any higher order of multimers. ATP depletion did not halt Gag assembly in the E. coli cells, and the addition of GroEL-GroES to in vitro reactions did not facilitate Gag assembly, indicating that conformational changes rather than protein refolding by chaperonins, induced at 37 °C, were solely responsible for the Gag assembly observed here. We suggest that Gag assembles to a capsid through the formation of the 60 S multimer, possibly a key intermediate of the assembly process, accompanied with conformational changes in Gag.

Received for publication, December 9, 2003 , and in revised form, April 27, 2004.

^* This work was supported by a Health Sciences Research Grant from the Ministry of Health, Labor and Welfare of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 81-3-5791-6129; Fax: 81-3-5791-6268; E-mail morikawa{at}lisci.kitasato-u.ac.jp.

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